The overall goal is to determine the biochemical and molecular processes that govern the differentiation of 3T3-L1 adipocytes. An important initial objective is to determine the mechanisms by which insulin-like growth factor-I (IGF-I) and/or insulin participate in the triggering of the adipocyte differentiation program and the modulation of the expression of adipocyte-specific gene products. When hormones and growth factors are removed from fetal calf serum the differentiation of 3T3-L1 cells into adipocytes becomes highly dependent on either physiological concentrations of IGF-I or a supraphysiological level of insulin. The unmasking of the striking effect of IGF-I and the acquisition of experience and expertise in recombinant DNA methodology in my laboratory over the past several years now afford us the opportunities to (a) probe and elucidate fundamental mechanisms in mammalian cell differentiation and (b) to obtain novel information and insights regarding a possible central biological role for IGF-I in the control of adipogenesis. We will approach these issues by executing the following specific aims: (1) Clone cDNAs corresponding to mRNAs that are induced at various stages of adipocyte development. (2) The cDNA clones will be used to determine the sizes and kinetics of accumulation of the IGF-I/insulin regulated mRNAs. (3) The mechanisms responsible for differentiation-dependent and IGF-I/insulin regulated accumulation of specific mRNAs will be determined. (4) IGF-I/insulin regulated genes will be characterized. (5) Cis-acting regulatory sequences that confer regulation by insulin/IGF-I will be identified and characterized by the construction of fusion genes, transfection, expression and mutagenesis. (6) IGF-I and insulin-binding studies will be performed to correlate the level of occupancy of specific IGF-I and insulin receptors with the degree and extent of regulation of specific genes.